In Vitro Biosynthesis of the [Fe]‐Hydrogenase Cofactor Verifies the Proposed Biosynthetic Precursors

Abstract In the FeGP cofactor of [Fe]‐hydrogenase, low‐spin FeII is in complex with two CO ligands and a pyridinol derivative; the latter ligates the iron with a 6‐acylmethyl substituent and the pyridinol nitrogen. A guanylylpyridinol derivative, 6‐carboxymethyl‐3,5‐dimethyl‐4‐guanylyl‐2‐pyridinol (3), is produced by the decomposition of the FeGP cofactor under irradiation with UV‐A/blue light and is also postulated to be a precursor of FeGP cofactor biosynthesis. HcgC and HcgB catalyze consecutive biosynthesis steps leading to 3. Here, we report an in vitro biosynthesis assay of the FeGP cofactor using the cell extract of the ΔhcgBΔhcgC strain of Methanococcus maripaludis, which does not biosynthesize 3. We chemically synthesized pyridinol precursors 1 and 2, and detected the production of the FeGP cofactor from 1, 2 and 3. These results indicated that 1, 2 and 3 are the precursors of the FeGP cofactor, and the carboxy group of 3 is converted to the acyl ligand.


Heterologous production and purification of HcgB and HcgC proteins
HcgB and HcgC from Methanococcus maripaludis were heterologously produced in Escherichia coli BL21(DE3) and purified using His-tag affinity chromatography using Ni 2+ -charged HiTrap chelating column (GE Healthcare) as described before. [1]

Heterologous production and purification of Strep-tagged [Fe]-hydrogenase apoenzyme from M. maripaludis
The E. coli strain harbouring the expression vector for production of Strep-tagged Hmd grew aerobically at 37 °C in 2 l LB medium supplemented with 50 µg/ml kanamycin with agitation with a magnetic stirrer (PTFE coated bar). When the optical density (OD) at 600 nm became 1, hmd gene expression was induced by addition of 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG, final concentration) and further cultivation for 46 h at 37 °C. The cells were harvested by centrifugation using a Beckmann JLA 10.500 rotor at 7,300 rpm at 4 °C for 20 min and stored until further use at −20°C. The frozen cells were suspended in 40 ml of 100 mM Tris-HCl pH 8 containing 150 mM NaCl, 2 mM dithiothreitol (DTT) and 5% (v/v) glycerol (Buffer A) and lysed by sonication using SONOPULS GM200 (Bandelin) with KE76 tip in ice water with a 50% cycle and 160 W (ten times for 1 min, with 1 min pauses). The cell debris and membrane particles were removed by ultracentrifugation using a Sorvall T647.5 rotor at 30,000 rpm at 4 °C for 30 min. The supernatant was applied to a 5 ml StrepTrap column equilibrated with buffer A. The column was washed with 10 column volumes (CV) of buffer A and then the apoenzyme was eluted by buffer A containing 2.5 mM desthiobiotin. The purified protein was desalted with buffer A by HiPrep 26/10 desalting column, concentrated, frozen in liquid N2 and stored at −75 °C until further use.
Step 2: Compound P2 (482 mg, 2.0 mmol) was dissolved in dry THF (5 mL) and cooled to −78 o C. Lithium diisopropylamide (LDA) (2 M in THF, 2.9 mL, 2.9 eq) was added slowly and the mixture was stirred at this temperature for 2 h. Then, dimethyl carbonate (216 mg, 1.2 eq) in THF (2 mL) was added dropwise. The reaction was stirred for another 30 min and quenched with water. The mixture was extracted with Et2O and purification by silica column chromatography using hexane/EtOAc as the eluent, product P3 was obtained as oil (100 mg, 17%). 1  Step 3: NaOH (615 mg, 5 eq) in H2O (20 mL) was added to the solution of compound P3 (920 mg, 3.1 mmol) in MeOH/THF (40 mL / 20 mL). The mixture was stirred at room temperature for 5 h until TLC showed no starting material remained. Aqueous HCl solution (~3 M) was added to adjust the pH to 1. All the volatile was then removed by vacuo. MeOH/EtOAc (30 mL / 30 mL) was added and filtered to remove some dissolved salts. After concentration and dryness, DCM (20 mL) was added. Then CF3COOH (20 mL) was added at 0 o C and the mixture was stirred at this temperature for 2 h. After stirring at room temperature for another 5 h, the mixture was concentrated. The residue was stirred with MeOH (2 mL) and Et2O (30 mL) for 30 min at room temperature. 2 was obtained as white solid after filtration (520 mg, 86%). 1

Synthesis and purification of 3,5,6-trimethyl-4-guanylyl-2-pyridinol (3')
Compound 3' was enzymatically synthesized from 2 and GTP by the reaction catalyzed by heterologously produced HcgB from M. maripaludis. The reaction solution containing 100 µM 2, 5 mM GTP/MgCl2 and 10 µM HcgB in 50 mM MOPS/KOH pH 7 was incubated for 48 h at 40 °C. The reaction was stopped by acidification with 40 µM acetic acid and then the protein was removed by centrifugation using Eppendorf MiniSpin plus at 14,500 rpm at room temperature for 20 min followed with filtration (0.22 µm pore size). The sample was concentrated by evaporation at 4 °C and applied to a HPLC system equipped with a Synergie 4 µm Polar-RP 80 Å column (Phenomenex, Aschaffenburg) with a water/methanol gradient. The fraction containing the sample were collected and concentrated by evaporation at 4 °C.

Purification of 6-carboxymethyl-3,5-dimethyl-4-guanylyl-2-pyridinol (3) from Methanothermobacter marburgensis
M. marburgensis was cultivated hydrogenotrophically in a medium with low nickel concentration using 10 l fermenter as described previously. [4] One hundred gram of M. marburgensis cells were suspended in 50 mM potassium phosphate buffer pH 7. A crude extract was prepared by sonication using SONOPULS GM200 (Bandelin) with KE76 tip in ice water with a 50% cycle and 160 W (six times for 8 min, with 7 min pauses). A protein fraction-containing [Fe]-hydrogenase was obtained by ammonium sulphate precipitation and dialysis as described elsewhere. [4] Until this step, the preparation was performed anaerobically under 95% N2/ 5% H2 atmosphere in an anaerobic chamber (Coy Laboratory Products, Grass Lake, MI). After dialysis, the FeGP cofactor was extracted from the partially purified [Fe]-hydrogenase fraction under air by addition of a mixture of 60% methanol, 1 mM 2-mercaptoethanol and 1% ammonia solution (final concentrations) under room light. Denatured protein was removed by ultrafiltration (10-kDa cut off) and the solution was dried by evaporation at 4 °C. After dissolving in water containing 1% NH3, the product was loaded on an anion exchange chromatography (5 ml HiTrap Q HP column). After wash of the column with 5 CV water containing 1% NH3, the adsorbed compounds were eluted with linear gradient of NaCl (0−1 M) in water containing 1% NH3. Compound 3 was eluted at 0.35 M NaCl concentration and further purified by HPLC using a Synergi 4m Polar RP 80A column (250 mm  4.6 mm, Phenomenex) with a water/methanol gradient. The purified 3 was finally concentrated by evaporation at 4 °C.

Cultivation of Methanococcus maripaludis
Methanococcus maripaludis ΔhcgB strain was cultivated in the 37 °C cultivation room using a modified medium [5] with sodium formate as substrate under 80% N2 / 20% CO2 with Tris as additional buffer component [6] in 5 l or 500 ml scale to an OD at 600 nm of 0.7-0.9. The actively growing cells were anaerobically harvested by a continuous-flow centrifuge (Heraeus 3049 continuous flow rotor at 15,000 rpm at 4°C), resuspended in medium again and sedimented by centrifugation (Beckmann JLA 10.500 rotor at 7,300 rpm and 4°C). The use of the culture medium for resuspension aimed to avoid lysis of the cells in low salt concentration buffer solutions. The cell pellets were finally anaerobically resuspended in a lysis buffer: 50 mM Tris/HCl pH 7.5, 5 mM MgCl2 and 2.5 U/ml DNaseI, to a final concentration of 0.5 g cells/ml buffer. The 1 ml aliquots were frozen in liquid N2 and stored until use at −20°C. The frozen samples were anaerobically thawed on ice. Unbroken cells and membrane particles were removed by ultracentrifugation using a Sorvall TFT-80.4 rotor at 35,500 rpm and 4°C for 1 h. This supernatant is designated as cell extract and used for in vitro biosynthesis assay (see below).

Proteome analysis
Cell pellets were lysed and reduced by tris(2-carboxyethyl)phosphine (TCEP) in the presence of deoxycholate (DOC) at 90 °C for 10 min. After that it was incubated at 25°C for 30 min in ammonium bicarbonate pH 8.2 iodoacetic acid (IAA) and then digested overnight at 30 °C with trypsin, MS approved (Serva). Before LC-MS analysis, samples were desalted using C18 microspin columns (Nest Group) according to the manufacturer's instructions. Dried and reconstituted peptides were then analyzed using liquidchromatography-mass spectrometry carried out on a Orbitrap Exploris 480 instrument connected to an Ultimate 3000 RSLC nano and a nanospray ion source (all Thermo Scientific). Peptide separation was performed on a reverse phase HPLC column (75 μm x 40 cm) packed in-house with C18 resin (2.4 μm; Dr. Maisch) with a 135 min gradient (formic acid / acetonitrile). MS data were searched against an in-house Methanococcus maripaludis S2 protein database using SEQUEST embedded into Proteome Discoverer 1.4 software (Thermo Scientific).

In vitro biosynthesis of FeGP cofactor
We prepared the sample solution in a 1.5 ml Eppendorf plastic cup under 95% N2/ 5% H2 atmosphere in an anaerobic chamber (Coy). Twelve µl of a mixture of 100 mM Fe(SO4)2(NH4)2, 100 mM DTT, 200 mM sodium dithionite, 500 mM MgCl2 and 630 mM 3 was added to 200 µl cell extract and then, 5 µl of 250 mM S-adenosyl methionine, 2 µl 250 mM ATP and lastly 5 µl 500 mM heterologously produced Hmd apoenzyme from M. jannaschii were added. Addition of the Hmd apoenzyme in the earlier points made precipitates. The solution was transferred to a vial with a rubber stopper containing 50% H2 /50% CO or otherwise described atmosphere. The solution was incubated at 40 °C for 1 h or at room temperature for at least 3 h and the activity of [Fe]-hydrogenase was determined. The enzyme activity measurements were performed mostly in duplicates. The data for Figure 2a and 3a are triplicates. The activity was variable between the experiments using the cell extracts from different batches of the culture; therefore, the data sets shown in each panel were obtained using the same cell extract.
[Fe]-hydrogenase activity assay [Fe]-hydrogenase was determined by photometrically measuring the conversion of methylene-tetrahydromethanopterin (methylene-H4MPT) to methenyl-H4MPT + in 120 mM potassium phosphate buffer at pH 6 containing 1 mM EDTA in 1 ml cuvettes (1 cm light pass) in a total volume of 0.7 ml assay solution in the presence of 20 M methylene-H4MPT under 100% N2. [4] Methylene-H4MPT was prepared as described previously by spontaneous reaction of formaldehyde with H4MPT. [4] Increase rate of the absorbance at 336 nm was recorded and the activity was calculated using the extinction coefficient of methenyl-H4MPT + (336 nm = 21.6 mM -1 ·cm -1 ). One U was defined as the activity producing one mol of methenyl-H4MPT from methylene-H4MPT per min. Activities are given as U/mg protein in the assay. The protein concentration was assayed using Bradford method using the dye solution ROTI®Quant (Carl Roth) using bovine serum albumin (Bio-Rad Laboratories) as standard. The range of the protein concentrations in the assay was between 15-25 mg/ml.

Preparation of the FeGP cofactor from in vitro biosynthesis solution for mass spectrometric analysis
To obtain the purified FeGP cofactor for high resolution Orbitrap mass spectrometer analysis, the standard in vitro biosynthesis assay was performed in the presence of 40 µM Strep-tagged Hmd apoenzyme from M. maripaludis in a volume of 5 ml. After completion of the in vitro biosynthesis assay, 1 U avidin (per ml of the in vitro solution) was added and then the solution was diluted twofold with 150 mM Tris-HCl pH 8 containing 100 mM NaCl. The reconstituted holo-Hmd was purified from this solution with the same protocol as for the purification of Strep-tagged Hmd expressed in E. coli, except for that buffer B did not contain 5% glycerol. Just after the Streptag column purification, the FeGP cofactor was extracted from the purified enzyme by addition of 60% methanol, 1 mM 2-mercaptoethanol and 1% NH3 (final concentration) and incubated at 40 °C for 15 min, and then the extracted solution was filtrated (10 kDa cut off). The filtrate was concentrated by evaporation at 4 °C and the dried sample was dissolved in 400 l of 10 mM (NH4)2(CO3) pH 9 containing 1 mM 2-mercaptoethanol. The sample was filtered by a 0.22 µm filter before analysis by mass spectrometry.

Mass spectrometric analysis
Mass spectrometric determination of the FeGP cofactor was performed using a HRES-LC-MS. The chromatographic separation was performed on an Thermo Scientific Vanquish HPLC System using a polymer based ZICpHilic (Sequant, 150  2.1 mm, 5 µm, Merck) equipped with a 20  2.1 mm guard column of similar specificity at a constant eluent flow rate of 0.25 ml/min and a column temperature of 40 °C with eluent A being 10 mM ammoniumhydroxyde in water adjusted to a pH of 9.8 and eluent B being acetonitrile (Honeywell) The injection volume was 2 µl. The elution profile consisted of the following steps and linear gradients: 0 -3 min constant at 95 % B; 3 -10 min from 95 to 20 % B; 10 -20 min constant at 20 % B; 20 -20.1 min from 20 to 95 % B; 20.1 -30 min constant at 95 % B. Ionisation was performed using a high temperature electro spray ion source at a static spray voltage of 3300 V, Sheath gas at 35 (Arb), Auxilary Gas at 7 (Arb), and Ion transfer tube and Vaporizer at 300 and 275 °C. Full Scan measurements were conducted applying an orbitrap mass resolution of 240 000 without using quadrupole isolation in a mass range of 100 − 642. Data was saved in full profile mode. Targeted fragmentations measurements were performed at similar chromatography and ionisation settings, but using a quadrupole isolation of the target ion in a window of 0.4 m/z. Collision induced dissociation was performed in the ion routing multipole with a relative collision energy of 5 %. Fragments were detected using the orbitrap at a predefined mass resolution of 60 000 in the range between 100 and 640. For the analysis of the amount of incorporated 13 C in the [ 13 C]-CO labelling experiments the peak area of the extracted ion chromatogram of the calculated ion mass ± 5 ppm was integrated for each isotopologue and subsequently the natural isotope abundance of 13 C was subtracted using the IsoCor software. [7] Matrix assisted laser desorption ionization  time of flight mass spectrometry (MALDI-TOF-MS) was performed as described previously using a 4800 Proteomics Analyzer (Applied Biosystems/MDS Sciex). [1] Figure S1. Conversion of the UV-Vis spectrum upon dehydrogenation of methylene-H4MPT catalyzed by [Fe]-hydrogenase (Hmd) produced in the in vitro biosynthesis assay. (a) UV-Vis spectra of methylene-H4MPT (red), after the reaction with Hmd formed in the in vitro biosynthesis assay (cyan), after the reaction with Hmd purified from M. marburgensis as a positive control (violet), and the in vitro biosynthesis solution without methylene-H4MPT as a negative control (green). The peak observed at 336 nm indicates the presence of methenyl-H4MPT formed in the solutions. (b) Time-resolved UV-Vis spectrum changes by the reaction of Hmd produced in the in vitro biosynthesis assay. The spectra were recorded at each 10 s. The differential spectra were calculated by subtraction of the initial spectrum (Inset). Arrows indicate the direction of the change. The enzyme reaction assays were performed at the standard condition using 1 cm light pass cuvette in the presence of 40 M methylene-H4MPT. The reaction started by addition of 25 l 50-fold diluted in vitro biosynthesis solution.   . Preparation and characterization of non-natural guanylylpyridinol 3'. Compound 3' was guanylylated from the chemically synthesized pyridinol 2' by the HcgB catalyzed reaction. (a) HPLC chromatogram of the sample before (black) and after (green) the HcgB reaction. The spectra of 32 min peak (before reaction) and 28 min peak (after reaction) are identical to those of pyridinol 2 and guanylylpyridinol 3, respectively, [8] which indicated the conversion of pyridinol (2') to guanylylpyridinol (3') by the HcgB reaction (inset). The reaction product in the 28 min peak was used for in vitro biosynthesis.  In vitro biosynthesis activity obtained from 3'. In the abscissa, the precursors and the enzyme added in the assay are shown. As a positive control, in vitro biosynthesis activity obtained from 3 is shown. As a negative control, an assay without precursor was tested ().      PCR experiments confirmed that the genome of the hcgBhcgC strain contains the hcgF gene. HcgF was not detected by this proteome analysis. It was previously reported that deletion of hcgF resulted in a delay of growth, which was observed in the mutants lacking functional [Fe]-hydrogenase, while the phenotype was complemented by expression vector containing hcgF. [9] These experiments indicated that HcgF is functional in M. maripaludis.